I got a high background signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What went wrong?
Here are possible causes and solutions:
- Non-specific oxidation of substrate: Use less serum in the cell culture media; Note: Albumin can increase the auto-oxidation of Vargulin; Avoid repeated freezing and thawing of the sample.
- Control sample is contaminated: Use new sample; Change pipette tips after each well.
I am getting a high saturating signal with a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What could have happened?
This could be due to high luciferase expression. Here are some suggestions:
Reduce incubation time before collecting samples.
Decrease the integration time on the instrument.
Dilute the sample: for secreted Gaussia/Cypridina Luciferase, dilute the sample using media from the cell culture; for cell lysate, dilute the sample using lysis buffer
Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 µL per assay.
Why am I getting a low signal in lysate using a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay? Can you provide some suggestions?
Here are possible causes and solutions:
- Non-optimized lysis buffer used: Assay luciferase activity in the media to confirm good expression of luciferase; Use only the provided lysis buffer.
- Low luciferase expression: Lyse cells in smaller volume of 1X Cell Lysis Buffer; Use a different promoter or growth conditions to improve expression; Increase the integration time on the instrument; Scale up volume of sample and reagent per well.
I got a low signal in media when I used a Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. What happened?
Here are possible causes and solutions:
- Insufficient luciferase accumulation in media: Incubate cells for a longer time.
- Low luciferase expression: Use less media per well during the experiment;.Use a different promoter or growth conditions to improve expression;.Increase the integration time on the instrument;.Scale-up the volume of sample and reagent per well.
- Treatment interfered with cellular secretory pathway: Transfect cells with a plasmid for constitutive expression of Luciferase (i.e., with pTK or pCMV promoter); Determine if luciferase actively expresses in media without treatment. Add treatment; Determine if there is a corresponding drop in luciferase activity from the constitutively expressed plasmid.
I am getting no signal with my Gaussia/Cypridina/Renilla Luciferase Glow/Luciferase Flash assay. Why is this?
Here are possible causes and solutions:
- Low transfection efficiency: Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein); Verify plasmid DNA quality - use only transfection grade DNA; Use actively dividing, low passage cells; Use a different cell type.
- No or low promoter activity: Use conditions known for promoter activation; Incubate cells for a longer time; Change growth conditions to improve expression; Use a different promoter.
- Substrate auto-oxidized: Protect substrate from light and air; Maintain 100X Coelenterazine at -80 degrees C; maintain 100X Vargulin at -80 degrees C; Prepare new Coelenterazine Working Solution if used longer than 8 hours; Prepare new Vargulin Working Solution if used longer than 2 hours